ECSIT Is a Critical Limiting Factor for Cardiac Function

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ECSIT Is a Critical Limiting Factor for Cardiac Function ECSIT is a critical limiting factor for cardiac function Linan Xu, … , David J. Grieve, Paul N. Moynagh JCI Insight. 2021. https://doi.org/10.1172/jci.insight.142801. Research In-Press Preview Cardiology Metabolism Graphical abstract Find the latest version: https://jci.me/142801/pdf 1 2 ECSIT is a critical limiting factor for cardiac function 3 1 1 1 1 4 Linan Xu , Fiachra Humphries , Nezira Delagic , Bingwei Wang , 1 2 2 5 Ashling Holland , Kevin S. Edgar , Jose R. Hombrebueno , Donna 3 1 1 6 Beer Stolz , Ewa Oleszycka , Aoife M. Rodgers , Nadezhda 4 5 2 7 Glezeva , Kenneth McDonald , Chris J. Watson , Mark T. 5 2 2 8 Ledwidge , Rebecca J. Ingram , David J. Grieve and Paul N. 1,2,6* 9 Moynagh 10 1The Kathleen Lonsdale Institute for Human Health Research, Department of 11 Biology, Maynooth University, Maynooth, Co. Kildare, Ireland. 2Wellcome- 12 Wolfson Institute for Experimental Medicine, Queen's University Belfast, 13 Belfast, United Kingdom. 3Center for Biologic Imaging, University of Pittsburgh 14 Medical School, Pittsburgh, PA 15261, USA. 4Conway Institute, University 15 College Dublin, Belfield, Dublin, Ireland. 5Chronic Cardiovascular Disease 16 Management Unit and Heart Failure Unit, St Vincent's Healthcare Group/St 17 Michael's Hospital, County Dublin, Ireland 18 19 6Lead Contact: P.N.M. 20 *Correspondence: 21 Paul Moynagh (E: [email protected]; Tel: 00353-1-7086105)The Kathleen Lonsdale 22 Institute for Human Health Research, Department of Biology, Maynooth University, 23 Maynooth, Co. Kildare, Ireland. 24 The authors have declared that no conflict of interest exists 25 1 26 Abstract 27 ECSIT is a protein with roles in early development, activation of the transcription factor 28 NFB and production of mitochondrial reactive oxygen species (mROS) that facilitates 29 clearance of intracellular bacteria like Salmonella. ECSIT is also an important assembly factor 30 for mitochondrial complex I. Unlike the murine form of Ecsit (mEcsit), we demonstrate here 31 that human ECSIT (hECSIT) to be highly labile. In order to explore if the instability of hECSIT 32 affects functions previously ascribed to its murine counterpart, we created a novel transgenic 33 mouse in which the murine Ecsit gene is replaced by the human ECSIT gene. The humanised 34 mouse has low levels of hECSIT protein in keeping with its intrinsic instability. Whereas low 35 level expression of hECSIT was capable of fully compensating for mEcsit in its roles in early 36 development and activation of the NFB pathway, macrophages from humanised mice showed 37 impaired clearance of Salmonella that was associated with reduced production of mROS. 38 Notably, severe cardiac hypertrophy manifested in ageing humanised mice leading to 39 premature death. The cellular and molecular basis to this phenotype is delineated by showing 40 that low levels of human ECSIT protein leads to marked reduction in assembly and activity of 41 mitochondrial complex I with impaired oxidative phosphorylation and reduced production of 42 ATP. Cardiac tissue from humanised hECSIT mice also shows reduced mitochondrial fusion 43 and more fission but impaired clearance of fragmented mitochondria. A cardiomyocyte- 44 intrinsic role for Ecsit in mitochondrial function and cardioprotection is also demonstrated. We 45 also show that cardiac fibrosis and damage in humans correlates with low expression of human 46 ECSIT. In summary, our findings identify a new role for ECSIT in cardioprotection whilst also 47 generating a valuable new experimental model to study mitochondrial dysfunction and cardiac 48 pathophysiology. 49 Keywords: Cardiac Hypertrophy/ECSIT/Mitochondrial Complex I 50 2 51 Introduction 52 Toll-like receptors (TLRs) recognise pathogen-associated molecular patterns (PAMPs) 53 and respond by inducing the expression of pro-inflammatory proteins (1). The engagement of 54 TLRs by PAMPs leads to formation of receptor proximal signalling complexes consisting of 55 TIR domain-adaptor proteins, IRAK kinases and the E3 ubiquitin ligase TRAF6 (2). Activation 56 of the latter results in its auto-ubiquitination and formation of unanchored polyubiquitin chains, 57 leading to recruitment of Tak1 kinase and downstream activation of the transcription factor 58 NFB and MAP kinase pathways that drive inflammatory gene expression (3). Evolutionary 59 Conserved Signalling Intermediate in Toll pathway (Ecsit) was first described as a positive 60 regulator of NFκB by interacting with TRAF6 (4) and more recent reports also indicate its 61 interaction with Tak1 kinase (5) and NFB proteins (6). A mutant form of Ecsit, that strongly 62 activates NFκB, has been shown to drive hyperinflammatory disease (7). Other studies have 63 identified and characterised Ecsit as part of Complex I in the electron transport chain of 64 mitochondria (8-11). An N-terminal mitochondrial localisation sequence directs Ecsit to the 65 mitochondria to facilitate assembly of complex I. Furthermore in macrophages infected with 66 intracellular bacteria such as Salmonella Typhimurium (S. Typhimurium) Ecsit can interact 67 with TRAF6 to facilitate the juxtaposition of mitochondria and phagosomes and augment 68 reactive oxygen species (ROS) production to enhance bactericidal activity (12, 13). In 69 macrophages, Ecsit also mediates recruitment of phagosomes to damaged mitochondria to 70 promote removal of the latter by mitophagy (14). 71 Most analysis of Ecsit function has focused on the murine form of ECSIT (mEcsit) and at the 72 cellular level. Interestingly, across all human tissues, human ECSIT (hECSIT) is expressed at 73 its highest in heart but efforts to study the broader physiological role(s) of Ecsit, beyond innate 74 immunity, have been hampered by deletion of the Ecsit gene in mice resulting in early 75 embryonic lethality due to impaired BMP signalling during mesoderm formation (15). As part 76 of our efforts to study hECSIT, we noted early in our studies that the hECSIT protein is more 77 labile than its murine counterpart. To understand the physiological relevance of this differential 78 lability, humanised mice were generated in which the mEcsit gene was replaced by the hECSIT 79 gene. Tissues from humanised hECSIT mice displayed reduced levels of Ecsit but mice 80 survived and developed normally to adulthood. Macrophages from these humanised mice 81 showed normal activation of the NFB and MAP kinase pathways but reduced production of 82 mROS and impaired bacterial clearance in response to challenge with S. Typhimurium. Signs 83 of cardiac hypertrophy were apparent at a young age in the humanised hECSIT mice and 3 84 progressed to severe hypertrophy and heart failure in later life. This pathology was associated 85 with marked reduction in subunits and assembly of mitochondrial complex I with impaired 86 oxidative phosphorylation and reduced production of ATP. Cardiac tissue from humanised 87 hECSIT mice also showed reduced mitochondrial fusion and more fission but impaired 88 clearance of fragmented mitochondria. The effects of Ecsit depletion on cardiac hypertrophy 89 was also reproduced at a cell level in human cardiomyocytes confirming a cardiomyocyte- 90 intrinsic role for Ecsit in mitochondrial function and cardioprotection. In human cohorts, we 91 also demonstrate an inverse correlation between levels of hECSIT in heart tissue and cardiac 92 fibrosis and disease. This study adds further understanding to the recognised importance of 93 mitochondrial dysfunction being a major contributor to impaired cardiac functions (16). 94 Pathogenic mutations in core protein subunits and assembly factors of complex I, leading to 95 impaired complex I function, have been reported in a number of diseases including heart failure 96 and cardiomyopathy (17-19). This study now identifies hECSIT as a critical limiting factor for 97 mitochondria function and cardioprotection. 98 99 4 100 Results 101 Human ECSIT is more labile than murine Ecsit 102 As part of our efforts to study hECSIT, we initially noted intriguing differences in levels of 103 murine and human forms of ECSIT when we overexpressed myc-tagged forms of both proteins 104 in HEK293T cells (Supplemental Figure 1A, upper panels). The steady state expression levels 105 of hECSIT protein were consistently less than its murine counterpart. Whereas overexpression 106 of mEcsit was sufficient to induce expression of a co-transfected NFB-regulated reporter 107 gene, hECSIT was ineffective (Supplemental Figure 1A, lower panels). The inability of 108 hECSIT to activate NFB may be due to a potential functional difference from mEcsit or 109 alternatively its low level of expression. Indeed, mEcsit was more resistant to degradation than 110 hECSIT when cells were treated with the protein translation inhibitor cycloheximide 111 suggesting greater stability for mEcsit (Supplemental Figure 1B). Both proteins migrate as 2 112 immunoreactive bands with the slower migrating form of hECSIT being especially susceptible 113 to degradation. Both proteins were expressed at comparable levels in extracts from an 114 inducible prokaryotic expression system (Supplemental Figure 1C) but hECSIT was less stable 115 during purification to homogeneity with greater amounts of purified mEcsit being isolated from 116 similar scales of bacterial cultures (0.75 mg hECSIT versus 1.9 mg mEcsit from 1L culture, 117 Supplemental Figure 1D). We also compared the relative stabilities of both purified proteins 118 by characterising resistance to proteolytic processing by the non-specific protease Proteinase 119
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